Ion traps according to Paul comprise a ring electrode and two end cap electrodes, the ring electrode usually being supplied with the storage RF voltage, although other types of operation are possible. In the interior of the ion trap, ions can be stored in the essentially quadrupolar RF field. The ion traps can be used as mass spectrometers by ejecting the stored ions selectively according to their mass and measuring them using a secondary-electron multiplier. Several different methods for the ion ejection have been published; they will not be discussed further here.
The RF voltage on the ring electrode is very high, in customary ion trap mass spectrometers between 15 and 30 kilovolts (peak-to-peak). The frequency is around one megahertz. In the interior, a predominantly quadrupolar RF field is generated which oscillates with the RF voltage and drives the ions above a threshold mass towards the center, causing them to execute so-called secular oscillations in this field. The restoring forces in the ion trap are sometimes described by a so-called pseudo-potential which is determined by a temporal averaging of the forces of the real potential. In the center there is a saddle point of the oscillating real potential, which decreases quadratically, depending on the phase of the RF voltage, from the saddle point towards the ring electrode, and increases quadratically from the saddle point to the end cap electrodes (or the other way round in other RF phases).
Ion trap mass spectrometers have characteristics which make them of interest for many types of analyses. In particular, they can be used to isolate and fragment selected types of ions (so-called parent ions) in the ion trap. The spectra of these fragment ions are called “fragment ion spectra” or “daughter ion spectra” of the parent ions in question. It is also possible to measure “granddaughter ion spectra” as fragment ion spectra of selected daughter ions. Until now, the ions have been predominantly fragmented by a multitude of collisions with a collision gas, the oscillations of the ions to be fragmented being excited by a dipolar alternating field in such a way that the ions in the collisions can collect energy, a step which ultimately leads to the decay of the ions.
The ions can be either generated in the interior of the ion trap or introduced from outside. A collision gas in the ion trap ensures that the ion oscillations initially present are decelerated in the quadrupole RF field; the ions then collect as a small cloud in the center of the ion trap. The diameter of the cloud in normal ion traps is around one millimeter; it is determined by an equilibrium between the centripetal pseudo-force of the RF field (the restoring force of the pseudo-potential) and the repulsive coulomb forces between the ions. The internal dimensions of the ion trap are usually characterized by a separation of the end caps of around 14 millimeters; the ring diameter is around 14 to 20 millimeters.
A popular type of ionization of large biomolecules is the electrospray method (ESI=electro spray ionization), which ionizes ions at atmospheric pressure outside the mass spectrometer. These ions are then brought via inlet systems of a known type into the vacuum of the mass spectrometer and from there into the ion trap.
This ionization generates practically no fragment ions, the ions being essentially those of the molecule. With electrospray, multiply charged ions of the molecules do frequently occur, however. As a result of the lack of almost any fragment ion during the ionization process, the information from the mass spectrum is limited to the molecular weight; there is no information about internal molecular structures which can be used for the further identification of the substances present. This information can only be obtained by acquiring fragment ion spectra.
Recently, a particularly favorable method for the fragmentation of biomolecules, mainly peptides and proteins, has been developed in ion cyclotron resonance or Fourier transform mass spectrometry. It consists of allowing electrons to be captured by multiply positively charged ions, during which the ionization energy (more precisely: the proton attachment energy) released leads to the fragmentation of the usually chain-shaped molecules. The method is known as ECD (electron capture dissociation). If the molecules were doubly charged, one of the two fragments created remains as an ion. In this process, the fragmentation follows extremely simple rules (for specialists: there are predominantly c-cleavages and only a few a-cleavages and z-cleavages between the amino acids of a peptide), so that it is very simple to draw conclusions relating to the structure of the molecule from the fragmentation pattern. In particular, the sequence of peptides or proteins is easy to see from the fragmentation spectrum. The interpretation of these ECD fragment spectra is simpler than the interpretation of collision generated fragment spectra.
It is also possible to fragment triply or multiply charged ions in this way, but the method really shines in the case of doubly charged ions. If an electrospray ionization is applied to peptides, the doubly charged ions are also the most prevalent ions, as a rule. Electrospray ionization is a method of ionization which is particularly frequently used for biomolecules for the purpose of mass spectrometric analysis in ion traps.
For fragmentation by electron capture, the kinetic energy of the electrons must be very low, since otherwise there can be no capture. In practice, one offers electrons with an energy which lies just above the thermal energy of the electrons at room temperature. In the extremely strong magnetic fields of Fourier transform mass spectrometers this is very successful, because the electrons simply drift along the magnetic field lines until they reach the cloud of ions. A second energy regime between 3 and 30 electron volts leads to so-called “hot electron capture dissociation”, also a favorable dissociation method.
In electric RF ion traps according to Paul, it is difficult to create such an ion capture. As a rule, ion traps have perforations in the end caps through which the ions can enter and exit. In the case of internal ionization, the ionizing radiation is also introduced through this end cap perforation. An electron beam is usually used for this. The strongly oscillating RF field in the interior of the ion trap either accelerates the electrons in such a way that they rush through the trap volume with considerable energy or, alternatively, the electrons are turned back already at the entrance hole. These electrons are not particularly suitable for electron capture. Only for an extraordinarily short period of time, fractions of nanoseconds at the zero crossover of the high voltage, is there no field, and low energy electrons can reach the ion cloud with low energies. These few low energy electrons are in competition with very many more electrons which are accelerated to considerable energies, however; the fragmentation by high energy electron collision exceeds the fragmentation by electron capture many times over, thus making the fragment ion spectra useless.
In patent specification DE 100 58 706 C1 (U.S. Pat. No. 6,653,622), a method for ion trap mass spectrometers according to Paul has now been elucidated by which, in a simplest embodiment, the electrons are injected into the ion trap through an additional opening mounted in the ring electrode, the electron source being at such a high positive potential that it is equaled or exceeded by the oscillating potential of the center of the ion trap for only a very short period of time, only for a few nanoseconds in the maximum of the RF voltage. The electrons can reach the ion cloud only during these few nanoseconds, but they are decelerated to a mere fraction of their kinetic energy and are thus ideal for electron capture. At all other times, the electrons cannot reach the center of the ion trap at all because the potential of the center is more negative than the potential of the electron source and it repels the electrons, which are always negatively charged.
The deceleration of the ions in this case takes place en route from the ring electrode to the center, during which time the electrons must climb the saddle-shaped potential mountain between the two end caps (see FIGS. 3 and 4). The ion cloud is at the saddle point. The saddle potential focuses the electrons on the ion cloud in the plane formed by the beam axis of the electron beam and the z-axis, which passes through both end caps, (the “end cap plane”); electrons deviating laterally are forced back onto the correct path in the saddle channel again.
Unfortunately, there is no focusing of the electron beam in the other plane, the center plane of the ring (“ring plane”), instead, a defocusing occurs because the electrons here do not climb the potential of the center in a saddle, but rather on the outer shell of a rotation paraboloid. Only electrons which arrive exactly on the ideal line have a chance of climbing the mountain, but they also find themselves permanently in unstable equilibrium at this time and this causes them to immediately leave the ideal line each time there is the slightest perturbation. This defocusing has, until now, prevented the electrons reaching at the cloud.
The collision fragmentation in the ion trap usually occurs at an RF voltage of between one fifth and one third of the maximum voltage used for the scanning. This relatively high voltage is necessary in order to achieve sufficient energy transfer during the collisions. This voltage has the disadvantage, however, that fragment ions of low mass can no longer be held in the trap. It is therefore not possible to identify the complete sequence of a peptide because the small fragments with either one, two or three amino acids are lost.
The ion capture in the ion trap does not suffer from this disadvantage, if it can be created in the first place. This type of fragmentation can also take place at lower RF voltages so that fragment ions of low mass, i.e. those with one, two or three amino acids, can be held and detected in the trap.
For a fragmentation by electron capture, one option is that, after isolating doubly charged ions in the ion trap, an RF voltage of around 3 kilovolts (peak-to-peak), for example, is set, said voltage oscillating with a sine-shape at the ring in the potential range of −1.5 to +1.5 kilovolts against ground potential. The end cap electrodes are kept at ground potential. The center of the ion trap follows the ring voltage with approximately half of the ring electrode voltage if the inner radius of the ring electrode is 1.4 times the distance between the end cap electrodes, i.e. from around −750 to +750 volts. If the electron source is at a DC voltage potential of +750 volts, the electrons can only reach the center when the ring voltage in the voltage maximum is at +1.5 kilovolts and the center correspondingly at +750 volts. In this case, the electrons are accelerated outside the ion trap from the potential of the electron source (+750 V) to the potential of the ring electrode (+1.5 kV), thus receiving an energy of 750 electron-volts. In the interior of the ion trap, the kinetic energy of 750 electronvolts is decelerated to practically zero electron-volts again, because the center with the ion cloud is at the potential of +750 volts. At all other times, the center is at a more negative potential, the negative electrons are repelled.
Unfortunately, it has not yet proved possible to create the electron capture fragmentation in a quadrupole ion trap experimentally, because the electrons cannot reach the saddle point due to defocusing along the unstable potential increase in the plane of the ring.